international journal of hydrogen energy 34 (2009) 812–820

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/he

Hydrogen production characteristics of the organic

fraction of municipal solid wastes by anaerobic mixed
culture fermentation

Li Donga,b,*, Yuan Zhenhonga, Sun Yongminga, Kong Xiaoyinga, Zhang Yua,b

a
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, China
b
Graduate School of the Chinese Academy of Sciences, Beijing 100049, China

article info abstract

Article history: The hydrogen production from the organic fraction of municipal solid waste (OFMSW) by
Received 8 October 2008 anaerobic mixed culture fermentation was investigated using batch experiments at 37  C.
Received in revised form Seven varieties of typical individual components of OFMSW including rice, potato, lettuce,
12 November 2008 lean meat, oil, fat and banyan leaves were selected to estimate the hydrogen production
Accepted 13 November 2008 potential. Experimental results showed that the boiling treated anaerobic sludge was
Available online 13 December 2008 effective mixed inoculum for fermentative hydrogen production from OFMSW. Mechanism
of fermentative hydrogen production indicates that, among the OFMSW, carbohydrates is
Keywords: the most optimal substrate for fermentative hydrogen production compared with proteins,
OFMSW lipids and lignocelluloses. This conclusion was also substantiated by experimental results
Hydrogen of this study. The hydrogen production potentials of rice, potato and lettuce were 134 mL/
Anaerobic fermentation g-VS, 106 mL/g-VS, and 50 mL/g-VS respectively. The hydrogen percentages of the total gas
Mixed culture produced from rice, potato and lettuce were 57–70%, 41–55% and 37–67%.
ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights
reserved.

1. Introduction
The excessive use of fossil fuels is one of the primary causes of
global warming and acid rain, which have started to affect the
earth’s climate, weather condition, vegetation and aquatic
ecosystems. Considering the global environment, there is
a pressing need to develop non-polluting and renewable
energy source. As a sustainable energy source, hydrogen is
a promising alternative to fossil fuels. It is a clean and envi-
ronmentally friendly fuel, which produces water instead of
greenhouse gases when combusted. Furthermore, it has
a high energy yield (122 kJ/g), which is about 2.75 times greater
than that of hydrocarbon fuels, and could be directly used to
produce electricity through fuel cells [1,2]. Generally, there are
four available basic processes for the production of hydrogen
from non-fossil primary energy sources. These processes
include: (1) water electrolysis; (2) thermo-chemical processes;
(3) radiolytic processes; and (4) biological processes. For global
environmental considerations, biohydrogen production from
renewable organic waste represents an important area of
bioenergy production [3–5]. Biological hydrogen can be
generated by several ways. Hallenbeck and Benemann [6,7]
described the fundamentals of biological hydrogen produc-
tion: light-driven processes and dark fermentations. Nandi
and Sengupta reviewed the photosynthetic and fermentative
biological routes of hydrogen production [8]. Many studies

* Corresponding author. Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Research Center of Biomass Energy,
No. 1, Nengyuan Road, Wushan, Tianhe District, Guangzhou 510640, Guangdong, China. Tel.: þ86 20 8705 1423; fax: þ86 20 8705 7737.
E-mail address: lidong@ms.giec.ac.cn (L. Dong).
0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2008.11.031
 international journal of hydrogen energy 34 (2009) 812–820
have reported on the groundwork for biohydrogen production
systems through photosynthesis [9–11]. However, hydrogen
production by fermentative bacteria is technically simpler
than that by photosynthetic bacteria. Because it does not rely
on the availability of solar energy with large surface area and
transparency of the mixed liquor of the wastewaters stream
[12].
Gustavo et al. [13] reviewed fermentative hydrogen
production from various substrates by different inoculum.
These substrates not only included pure carbohydrates, such
as glucose [14–16], xylose [17], sucrose [18–20] and lactose [21],
but also molasses [22] and starch [23]. Besides, fruit waste [24],
palm oil mill effluent [25], food waste [26–28] and other organic
solid waste [29,30] was studied focusing on feasibility and
process parameters of fermentative hydrogen production.
Most of the inoculums for fermentative hydrogen produc-
tion were pure culture (such as Clostridium sp.). Clostridium sp.
is obligate anaerobes which are very sensitive to minute
amounts of dissolved oxygen. In order to grow Clostridium sp.
for hydrogen production, addition of expensive reducing
agents (such as L-cysteine) may be required, reducing the
feasibility in practical applications [31]. Therefore, some
researchers use a mixed culture as inoculum. This mixed
culture includes other fermentative hydrogen production
facultative anaerobes (such as Enterobacter sp.). Yokoi et al. [32]
have utilized a mixture of Enterobacter sp. and Clostridium sp. to
produce hydrogen under a reducing agent-free culture
medium and thereby lowering the cost of hydrogen produc-
tion. Treatments of sludge used as mixed inoculum (including
heating, chemical acidification, adding methanogens inhib-
itor, freezing and thawing, sterilizing and sonication) have
been investigated as a method for increasing hydrogen
production by altering the nature of the anaerobic microbial
communities [33,34]. Hydrogen-producing anaerobic bacteria
such as Clostridium species form endospores when unfavor-
able environmental conditions are encountered by bacterial
stresses (e.g., elevated temperature, chemicals, and radiation)
[35,36]. If the activities of non-spore-forming hydro-
genotrophic methanogens are inhibited by the dominance of
spore-forming hydrogen- producing bacteria, the fermenta-
tion may possess a significant capacity for the conversion of
organic wastes into hydrogen [14,20].
Selection of a proper pH is also crucial to enhance
hydrogen production, due to the effects of pH on hydrogenase
activity or metabolic pathways [37]. Some investigators [38,39]
reported that maximum hydrogen yield was determined at
a pH value of 5.5. Whereas Lee et al. [40] reported that the
maximum hydrogen yield was achieved at initial pH 9.0.
Cheong et al. [41] found maximum hydrogen yield under
culture conditions of initial pH 7. These conflicting results
seem to be due to the initial pH of the culture without main-
tained buffering capacity for preventing a decrease of pH.
Little information is available on the biohydrogen produc-
tion characteristics and mechanism from the organic fraction
of municipal solid waste (OFMSW) by mixed culture. As
a major burden to the environment, the generation of
municipal solid waste (MSW) amounts to 170 million tons in
2006 in China, and this number is growing by 6% per year [42].
Nearly 60% of MSW is organic fraction such as kitchen waste,
waste paper and urban greening waste. To treat OFMSW by
813
fermentative hydrogen production, not only alleviates conflict
between energy supply and demand in a certain extent, but
also improves economic feasibility for MSW treatment.
The purpose of this study is to investigate the character-
istics and mechanism of biohydrogen production from the
individual OFMSW by batch experiments using mixed culture
as inoculum. For this purpose, seven varieties of typical
organic materials were selected and their hydrogen produc-
tion characteristics were studied. These materials included
rice, potato, lettuce, lean meat, oil, fat and Banyan leaves
which represent natural starches, cellulose, protein, vegetable
lipid, animal lipid and lignocellulose materials widely existed
in OFMSW.
2. Materials and methods
2.1. Seed microorganisms
The seed used in this study was heat treated anaerobic sludge.
The sludge was originally obtained from an swine manure
anaerobic digester and acclimatized with kitchen waste for
2 month at 37  C. Firstly, the sludge was introduced into the
anaerobic reactor, then the kitchen waste was added once
a week and the adding amount increased step by step. Prior
to use, the sludge was sieved to remove bone, sand and
other coarse matters. Thereafter, the sludge was boiled for
15 min to inactivate the hydrogentrophic methanogens and to
enrich the hydrogen-producing bacteria. The pH, ammonia
nitrogen, alkalinity, volatile fatty acids (VFAs) and volatile
suspended solid (VSS) were 9.2, 230 mg/L, 860 mg/L, 119 mg/L
and 3750 mg/L.
2.2. Experimental substrate
The materials used in this study are given in Table 1. All feed
amount of substrate was 8.0 g (calculated by VS) except meat
of 5.0 g in order to avoid ammonia inhibition [43].
2.3. Experimental setup and procedure
A 500 ml serum bottle used as a reactor was placed in the
water bath at 37  1  C. The substrate and 200 mL inoculum
were added into the bottle. For the nitrogen-poor substrates
(including rice, potato, oil, fat and banyan leaves), 5 ml
NH4HCO3 solution of 200 g/L was added to supplement
nitrogen source. Total substrate concentrations in reactor
were adjusted to 20 g-VS/L by adding distilled water except
meat of 12.5 g-VS/L. The initial pH was adjusted to 5.5 by
adding 2 mol/L HCl or 2 mol/L KOH in this study. The head-
space of the reactor was filled with pure N2 to assure the
anaerobic condition. Mixing was conducted twice a day
manually. The anaerobic digestion was finished until no gas
was produced. Each experimental condition was carried out in
duplicate.
2.4. Analytical methods
Total solid (TS), volatile solid (VS), ammonia nitrogen and
alkalinity were determined using standard techniques [44].
814 international journal of hydrogen energy 34 (2009) 812–820

Table 1 – Characteristics of substrates

Substrate TS (g) Heat value (J/g TS) VS (%TS) VS (g) [C] (%TS) [H] (%TS) [O] (%TS) [N] (%TS) [C]/[N]

Rice 8.0 17053 99.5 8.0 42.63 5.74 50.06 0.89 47.7
Potato 8.0 16324 99.5 8.0 41.36 5.59 51.16 1.17 35.4
Lettuce 9.5 16669 84.6 8.0 42.12 4.84 33.66 3.26 12.9
Lean meat 5.3 24077 94.9 5.0 50.97 6.10 23.04 13.11 3.9
Oil 8.0 38196 100 8.0 76.40 7.90 12.90 0.01 6967
Fat 8.0 38917 100 8.0 78.20 10.0 9.00 0.01 8279
Banyan leaves 9.3 17563 85.9 8.0 45.34 4.98 34.48 0.36 125.7

Heat values were determined by WGR-1 heat value

analyzer. Elementary analysis was determined by Vario EL
element analyzer. The pH was determined by pHS-3C pH
meter.
Biogas production was measured by the displacement of
saturated brine solutions. The gas volumes were corrected to
a standard temperature (0  C) and pressure (1 atm) (STP). The
compositions of biogas (H2, CH4 and CO2) were analyzed by
a gas chromatograph (Agilent 6890) equipped with a thermal
conductivity detector (TCD) and a 2 m stainless column
packed with Porapak Q (50/80 mesh). The operational
temperature at the injection port, the column oven and the
detector were 100  C, 70  C and 150  C respectively. Argon was
used as a carrier gas at a flow rate of 30 mL/min.
Liquid samples were centrifuged with 8000 rpm at 0–4  C
and filtrated with 0.45 mm filter. The concentrations of VFAs
and alcohols were determined using a gas chromatograph
(Agilent 6820) equipped with a flame ionization detector (FID).
The packed capillary column (DB-FFAP) was 30 m  0.25 mm
with inner diameter 0.25 mm. Nitrogen was used as a carrier
gas at a flow rate of 30 mL/min and split ratio was 1:50. The
operational temperature of injection port and detector were
250  C and 300  C. The initial temperature of oven was 40  C
for 5 min, then increased to 140  C at rate of 10  C/min and
maintained for 1 min, subsequently increased to 250  C at rate
of 5  C/min and maintained for 3 min. The VFAs and alcohols
analyzed included acetate, propionate, butyrate, isobutyrate,
valerate, isovalerate, methanol, ethanol, propanol and
butanol.
The hydrogen production efficiency was evaluated using
the hydrogen content (HC) in the biogas, hydrogen yield (the
ultimate hydrogen production per gram VS, HY, mL/g-VS),
volumetric hydrogen production rate (the hydrogen produc-
tion from 1 L culture per day, VHPR, mL/(Lculture  d)) and
specific hydrogen production rate (the hydrogen production
from 1 g of dry biomass per day, SHPR, mL/(g-VSS  d)). The
modified Gompertz equation was used to describe the prog-
ress of cumulative hydrogen production obtained from the
batch experiments [45]. Using the cumulative hydrogen
production data to fit the modified Gompertz equation, the
maximum hydrogen production rates were estimated.
  
Rm  e
HðtÞ ¼ P  exp  exp ðl  tÞ þ 1 ; (1)
P
Where H (t) is cumulative hydrogen production (mL), P
hydrogen production potential (mL), Rm maximum hydrogen
production rate (mL/d), e ¼ 2.71828, l lag-phase time (d) and t
time (d).
3. Results and discussion
3.1. Hydrogen production potential for different
substrates
Fig. 1 shows the cumulative hydrogen production and
hydrogen content. For the carbohydrates substrate (including
rice, potato and lettuce), gas production began immediately
after the inoculation. The produced gas was found to consist of
hydrogen and carbon dioxide. Methane was never detected
during the incubation of each substrate. During the first
2 days, cumulative hydrogen production rapidly increased and
then reached a plateau for each substrate. Hydrogen concen-
tration increased sharply and reached a maximum value of
70% at 3 days, 55% at 1 day, and 67% at 1.5 days for rice, potato
and lettuce respectively. But they declined to 26%, 22% and
17% finally. The decrease in hydrogen concentration was likely
to be associated with the consumption of hydrogen. However,
no methanogenesis was observed throughout the experiment
due to the inhibition of methanogenic activity by thermal
treatment. This implies that hydrogen might be utilized by
some other microorganisms, such as homoacetogens [46,47].
The hydrogen yields for rice, potato and lettuce were 134 mL/
g-VS, 106 mL/g-VS and 50 mL/g-VS respectively. It is notice-
able that there was a lag time of about 15 h for rice. For the
protein substrate, only 18 mL gas was produced during the
hydrogen production fermentation of lean meat. No hydrogen
was detected during the incubation with carbon dioxide as
main gas component. For the lipid substrate (including oil and
fat), the gas production was less than that using carbohydrates
as the substrate. A hydrogen yield of 6.25 mL/g-VS indicates
that about 0.1% of oil was converted to hydrogen based on
COD. Unpredictably, no hydrogen was produced with fat as
a substrate. For the lignocellulose substrate, a hydrogen yield
of 1.75 mL/g-VS indicates that less than 0.1% of banyan leaves
was converted to hydrogen based on COD.
Fig. 2 shows the modified Gompertz equation curves fitted
using the cumulative hydrogen production data for rice,
potato and lettuce. The hydrogen production potentials, the
maximum hydrogen production rates and lag-phase time
were estimated using modified Gompertz equation. The
results are given in Table 2. The hydrogen production poten-
tial of lettuce was less than that of rice and potato, since
cellulosic carbohydrate was more recalcitrant to degradation
compared with amylaceous carbohydrate.
Several studies have also reported hydrogen production
from various carbohydrates by pure culture and mixed
 international journal of hydrogen energy 34 (2009) 812–820 815

80 2000 80
cummulative gas production (mL)

cummulative gas production (mL)

2000 Rice Potato

hydrogen content (% v/v)

hydrogen content (% v/v)
1600
60 60
1500
1200

40 40
1000
800
total gas total gas
500 20 20
hydrogen 400 hydrogen
hydrogen content hydrogen content

0 0 0 0
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
time time
1000 400 80

cummulative gas production (mL)

80
cummulative gas production (mL)

Lettuce Lean meat

350

hydrogen content (% v/v)

hydrogen content (% v/v)

800
60 300 60
total gas 250
600 hydrogen total gas
hydrogen content 40 200 40
hydrogen
400 150 hydrogen content

20 100 20
200
50

0 0 0 0
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
time time

400 80 400 80
cummulative gas production (mL)

cummulative gas production (mL)

Oil Fat
hydrogen content (% v/v)

hydrogen content (% v/v)

300 60 300 60

200 total gas 40 200 40

hydrogen
hydrogen content total gas
100 20 100 hydrogen 20
hydrogen content

0 0 0 0
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
time time

200 80
cummulative gas production (mL)

Banyan leaf
hydrogen content (% v/v)

150 60

100 40
total gas
hydrogen
50 hydrogen content 20

0 0
0 1 2 3 4 5 6 7
time

Fig. 1 – Cumulative hydrogen productions and hydrogen contents.

816 international journal of hydrogen energy 34 (2009) 812–820

cumulative hydrogen production (mL)

1200 acetate were the main intermediate metabolites for rice,
potato and lettuce, accounting for more than 75% (w/w) of the
1000 total VFA and alcohols, suggesting a butyrate-type fermenta-
tion in this experiment. The results also suggest that the
800 mixed culture used in this study possessed clostridial char-
rice acteristics as evidenced by the typical hydrogen/acid-
600 potato producing phases.
lettuce The VFAs can be stimulatory, inhibitory or even toxic to
400 the fermentative bacteria depending on their concentrations.
A low level of VFAs may have no effect or a stimulatory effect
200 on hydrogen production. However, at a high level, VFAs can
lead to severe inhibition on hydrogen fermentation. It is
0 presumed that the VFAs in their undissociated forms can
0 1 2 3 4 5 6 7
time (d) freely permeate the bacteria cell membrane [52]. If the
undissociated VFAs are pumped into the culture, they will
Fig. 2 – Modified Gompertz equation fitting curves for rice, penetrate the plasma membrane and dissociate depending on
potato and lettuce. the pH inside cell. Thus, the pH inside the cell will be lowered.
To prevent unfavorable physiological conditions within the
bacteria cell, excess energy must be used to pump ions, e.g.
cultures in batch, continuous, and cell immobilization reac- potassium ions, from the culture. Therefore, the energy used
tors. Table 3 lists the results of these studies including HY, for bacteria growth is reduced and the bacteria growth rate
VHPR, SHPR and HC for comparison. Generally, the unit of will be lowered [53]. On the other hand, if a high level of
hydrogen yield was mol H2 / mol substrate or mL H2 / mol dissociated VFAs is present in the culture, the ionic strength
substrate which were reported in previous studies when in solution will increase. Such an increase can result in cell
substrate was pure carbohydrate (such as glucose and lysis. The effects of VFAs on the fermentative bacteria are
sucrose). But it is difficult to determine the molar amount of associated with the buffer capacity (such as pH and alkalinity
complex mixture, so in this paper, the units of hydrogen yields values). One of the criteria for judging fermentation stability
are unified to mL/g-VS at standard temperature (0  C) and is the ratio of VFAs/alkalinity. There are three critical values
pressure (1 atm) (STP). for this [54]:
As shown in Table 3, hydrogen yield is dependent on
substrate, seed, reactor type and culture conditions. More- <0.4 fermentation should be stable;
over, these factors are interactional, such as temperature, pH 0.4–0.8 some instability will occur;
and substrate concentration [19]. In general, the hydrogen >0.8 significant instability.
yield of cellulosic carbohydrate (such as microcrystalline
cellulose and lettuce) was less than pure and amylaceous In this study, the ratio of VFA/alkalinity at the end of
carbohydrate, because hydrogen production from them fermentation for rice, potato and lettuce substrate were far
requires hydrolysis reaction which requires long time. more than 0.8 which resulted in severe inhibition. It can be
concluded that the removal of VFAs is required in order to
3.2. Production of soluble intermediate metabolites produce hydrogen continuously. On the other hand, the
energy contained in VFAs and alcohols should be further
The production of hydrogen accompanied VFAs and alcohols recovered. The hydrogen production fermentation followed
production during the anaerobic fermentation of organic by methane fermentation or photo-fermentation can realize
substrate. The concentration and distributions of VFAs have this objective. During the following step, organic acids are
been used as a useful indicator for monitoring hydrogen converted to methane by methanogen or to hydrogen by
production [48]. The VFAs and alcohols concentrations at the photosynthetic bacteria.
end of fermentation were given in Table 4. The main inter-
mediate metabolites were VFAs with negligible alcohols. The
VFAs concentrations were in line with hydrogen yields since
VFAs were byproducts of hydrogen production fermentation.
Table 2 – Modified Gompertz equation parameters for
From the results of intermediate metabolites, it is concluded rice, potato and lettuce
that the degradation of meat, oil, fat and banyan leaves were
Substrate P Psa Rm Rmsb l (d) R2 T90c
limited. The production of VFAs lowered the pH for the rice, (mL) (mL/ (mL/d) [mL/ (d)
potato, lettuce, oil and fat. Whereas the fermentation of meat g-VS) (g-VS-d]
increased pH since the degradation of meat produced
Rice 1056 132 896 112 0.6 0.9990 1.9
ammonia. The same phenomenon was observed during the
Potato 816 102 744 93 0.2 0.9979 1.5
fermentation of banyan leaves, it was likely some alkalescent Lettuce 384 48 392 49 0.1 0.9960 1.3
compounds were produced. However it is required to be
warranted. a The hydrogen production potential per gram substrate.
b The maximum hydrogen production rate per gram substrate.
The concentrations of dominating VFAs and their fraction
c The time for cumulative hydrogen production achieving 90% of P.
in SMP are given in Table 5. Among them, butyrate and
 international journal of hydrogen energy 34 (2009) 812–820 817

Table 3 – Comparison of hydrogen production efficiency from various substrate by different seed
Substrate Seed Reactor type and HY VHPR SHPR HC Reference
(concentration) culture conditions (mL/g-VS) [mL/ [mL/ (%)
(Lculture  d)] (g-VSS  d)]

Glucose (10 g-VS/L) Heat treated anaerobic Serum bottles, 37  C, 217 – 1483 42 [49]
sludge from sewage initial pH 6.0
treatment plant
Sucrose (30 g-VS/L) Heat treated sludge from 5 L agitated fermenter, 143 4824 600 – [50]
UASB digester 35 C, controlled pH 5.5
Microcrystalline Heat treated sludge from Serum bottles, 37  C, 36 350 303 50 [51]
cellulose (25 g-VS/L) anaerobic high-solids initial pH 7.0
digester
Jackfruit peel (33 g-VS/L) Heat treated microflora Anaerobic contact 198 – – 55 [24]
from cow dung filter, room temperature,
controlled pH 5.3
Food waste (10 g-VS/L) Thermophilic acidogenic Serum bottles, 55  C, 70 – 456 69% [26]
culture form CSTR controlled pH 5.5
Cabbage (30 g TS/L) Heat treated sludge from Serum bottles, 37  C, 62 – – 55% [30]
anaerobic digester initial pH 7.0
Carrot (20 g TS/L) Heat treated sludge from Serum bottles, 37  C, 71 – – 47% [30]
anaerobic digester initial pH 7.0
Rice (40 g TS/L) Heat treated sludge from Serum bottles, 37  C, 96 – – 46% [30]
anaerobic digester initial pH 7.0
Rice (20 g-VS/L) Boling treated sludge from Serum bottles, 37  C, 134 2240 1990 57–70 This study
anaerobic digester initial pH 5.5
Potato (20 g-VS/L) Boling treated sludge from Serum bottles, 37  C, 106 1860 1650 41–55 This study
anaerobic digester initial pH 5.5
Lettuce (20 g-VS/L) Boling treated sludge from Serum bottles, 37  C, 50 980 870 37–67 This study
anaerobic digester initial pH 5.5

3.3. Biohydrogen production characteristic of It is favorable thermodynamically using carbohydrate

components of OFMSW
3.3.1. Carbohydrates
Fermentative hydrogen production usually proceeds from the
anaerobic glycolytic breakdown of sugars. The majority of
microbial hydrogen production is driven by the anaerobic
metabolism of pyruvate, formed during the catabolism of
various substrates. Acetate and butyrate are formed during
hydrogen -producing fermentations.
substrate to produce hydrogen by anaerobic fermentation.
In theory, acetate fermentation is capable of generating
4 mol H2 /mol glucose. This is the so-called ‘‘Thauer limit’’ [55].
Yield of hydrogen by butyrate fermentation carried out by some
clostridia are lower than that of acetate fermentation. Several
study results indicated that actual hydrogen yields were lower
than 4 mol that were theoretically possible, typically ranging
from 0.5 to 2.5 mol H2/mol hexose. In fact, the intermediate
metabolites often are a mixture of acetate and butyrate:

Glucose þ 2H2 O/2Acetate þ 2CO2 þ 4H2 DG0 ¼ 206:3 kj=mol
0
Glucose/Butyrate þ 2CO2 þ 2H2 DG0 ¼ 254:8 kj=mol
4Glucose/2Acetate þ 3Butyrate þ 8CO2 þ 8H2 (4)
(2) For this fermentation, the yield is 2 mol H2/mol glucose.
Approximately 50% of clostridia isolated to date carry out this
(3) mixed acids fermentation. Other fermentation pathways were

Table 4 – Final pH, VFAs, Alcohols, NH3-N, and Alkalinity at the end of fermentation
Substrate Final pH VFAsa (mg/L) Alcoholsb (mg/L) SMPc (mg/L) NH3-N (mg/L) Alkalinityd (mg/L) VFAs/alkalinity

Rice 4.56 14405  79 198  18 14603  98 810  16 1828  30 7.88

Potato 4.81 5288  49 473  20 5761  69 780  7 2944  14 1.80
Lettuce 5.14 5013  36 182  8 5195  45 850  13 2287  20 2.19
Meat 5.74 2324  11 42  10 2366  21 1600  43 4385  25 0.53
Oil 5.48 2212  10 118  16 2330  25 420  17 4659  54 0.47
Fat 5.28 1019  8 106  10 1125  18 510  13 4291  74 0.24
Banyan leaves 5.72 2181  16 141  4 2322  20 730  21 5108  47 0.43

a Including acetate, propionate, butyrate, i-butyrate, valerate, i-butyrate.

b Including methanol, ethanol, propanol and butanol.
c SMP ¼ VFAs þ Alcohols.
d Total alkalinity calculated by CaCO3.
818 international journal of hydrogen energy 34 (2009) 812–820

Table 5 – Distribution of intermediate metabolites

Substrate HAc (mg/L) HPr (mg/L) HBu (mg/L) EtOH (mg/L) HBu/HAc
a
Rice 7098  45 48.6% 2795  36 19.1% 3899  39 26.7% 145  11 1.0% 0.55
Potato 2906  32 50.4% 193  15 3.4% 2179  40 37.8% 426  55 7.4% 0.75
Lettuce 3416  39 65.8% 280  16 5.4% 1312  36 25.3% 86  9 1.7% 0.38
Meat 744  24 31.4% 462  24 19.5% 744  27 31.4% 42  12 1.8% 1.00
Oil 196  15 8.4% 163  20 7.0% 1789  18 76.8% 45  7 1.9% 9.13
Fat 860  21 76.4% 152  19 13.5% 76 0.6% 54  15 4.8% 0.01
Banyan leaves 1643  19 70.8% 223  11 9.6% 301  22 13.0% 71  6 3.1% 0.18

a % are percentage of individual HAc, HPr, HBu and EtOH in SMP.

found in sacchrolytic clostridia, such as the production of
propionate by Clostridium arcticum, succinate by Clostridium
coccoides, and lactate by Clostridium barkeri.
3.3.2. Lipids
Glycerol and long chain fatty acids (LCFA) are mainly
produced from anaerobic hydrolysis of lipids. Glycerol could
be a substrate for hydrogen production and for solvent
production with saccharolytic clostridia [56]. It is apparent
that glycerol may be suitable substrate for solvent production
rather than hydrogen production. The degradations of LCFA
(b-oxidation) are thermodynamically unfavorable reactions
unless the hydrogen partial pressure is maintained to an
extremely low level:
n  LCFA/ðn  2Þ  LCFA þ 2Acetate þ 2H2 DG0
¼ þ48 mj=mol (5)
PyruvateþAcetateþ2H2 /þCO2 þH2 O DG0 ¼ 95:4 kj=mol (6)
It only proceeds at very low partial pressures of hydrogen, below
103 atm, when the free energy change is negative. This low
partial pressure is realized in some natural anaerobic digestions
where the produced hydrogen is consumed timely by hydro-
genotrophic methanogens permits the reaction thermody-
namically favorable. But hydrogenotrophic methanogens are
absence in hydrogen production reactor where extremely low
hydrogen partial pressure can not be maintained. Moreover,
LCFA are one of the inhibitors for anaerobic bacteria [57]. They
adhere to cell wall of bacteria which restricts the transportation
of nutrition. Therefore, it may be difficult to produce hydrogen
from long chain fatty acids. Because a large portion of chemical
oxygen demand (COD) of lipid is converted to long chain fatty
acids during the hydrolysis reaction, even if glycerol could have
high hydrogen production potential, hydrogen production from
lipid may not be high. In fact, hydrogen production yields of oil
and fat were very low in this study.
3.3.3. Proteins
Protein is hydrolyzed to various amino acids by extracellular
enzymes. There are three types of amino acid degradation
reactions under anaerobic condition [55]:
1) Stickland reaction
Alanine þ 2glycine/3acetate þ 3NH3 þ CO2 (7)
2) Oxidative deamination from sole amino acid
Leucine þ 3H2 O/isovalerate þ HCO þ
3 NH4 þ 2H2 DG0
¼ þ4:2 kj=mol (8)
3) Reductive deamination from sole amino acid
Glycine þ H2 /acetate þ NH3 DG0 ¼ 77:8 kj=mol (9)
All degradation of amino acids involves production of volatile
fatty acids and ammonia. The concentration of ammonia
produced correlated with the amount of amino acids
(proteins) degraded. Therefore, the degree of protein degra-
dation can be known by observing ammonia concentration. In
this study, when lean meat was used as a substrate of
hydrogen production fermentation without NH4HCO3 added,
ammonia was produced and the concentration was higher
than that of other substrate fermentation with NH4HCO3
added. It can be concluded that the lean meat was degraded
partially. An 18 mL gas also testified this conclusion although
the gas was carbon dioxide without hydrogen produced. There
are some reasons why hydrogen was not produced. Firstly,
hydrogen is not produced by Stickland reaction. Moreover,
approximately 90% of amino acids degradation is carried out
by Stickland reaction [58]. Secondly, oxidative deamination
from sole amino acid is thermodynamically unfavorable
reactions unless the hydrogen partial pressure is maintained
to an extremely low level. Thirdly, amino acids such as
glycine, which was degraded by reductive deamination can
consume hydrogen as an electron donor [58]. Therefore, even
if hydrogen is produced from oxidative deamination, the
hydrogen can be utilized by reductive deamination. From the
above, it may be difficult to produce hydrogen from substrate
which includes a large quantity of protein.
3.3.4. Lignocelluloses
Cellulose, hemicellulose and lignin compose lignocellulose
which has a complicated structure. For untreated natural
lignocellulose materials, it is difficult to degrade. In most
cases, the cellulose fibers are embedded in a matrix of other
structural biopolymers, primarily hemicelluloses and lignin.
Complicated structure feature limits the rate and extent of
hydrolysis [59]. Therefore, it is very difficult to produce
hydrogen from lignocellulose substrate unless suitable
pretreatment is adopted.
4. Conclusion
1) Boiling treated anaerobic sludge is an effective mixed inoc-
ulum for fermentative hydrogen production from OFMSW.
2) Among the OFMSW, carbohydrates is the most optimal
substrate for fermentative hydrogen production compared
with proteins, lipids and lignocelluloses.
 international journal of hydrogen energy 34 (2009) 812–820
3) The hydrogen production potentials of three individual
carbohydrates were measured: rice 134 mL/g-VS, potato
106 mL/g-VS, and lettuce 50 mL/g-VS. The hydrogen percent-
ages of the total gas produced from rice, potato and lettuce
were 57–70%, 41–55% and 37–67%, respectively.
4) In order to produce biological hydrogen continuously,
the intermediate metabolites (including VFAs and alcohols)
should be removed timely to avoid inhibition. On the other
hand, intermediate metabolites should be utilized by a suit-
able secondary process step to increase the energy yields of
the waste. The methane fermentation or photo-fermentation
is suggested as secondary step to produce methane or
hydrogen.
Acknowledgment
This work was financially supported by the Guangdong
Science and Technology Program (No. 0711031100011).
references
[1] Ramachandran R, Menon RK. An overview of industrial uses
of hydrogen. International Journal of Hydrogen Energy 1998;
23(7):593–8.
[2] Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike T.
Enhancement of hydrogen production from glucose by
nitrogen gas sparging. Bioresource Technology 2000;73(1):
59–65.
[3] Momirlan M, Veziroglu T. Current status of hydrogen energy.
Renewable and Sustainable Energy Reviews 2002;6(1–2):141–79.
[4] Dunn S. Hydrogen futures: toward a sustainable energy
system. International Journal of Hydrogen Energy 2002;27(3):
235–64.
[5] Barretoa L, Makihiraa A, Riahia K. The hydrogen economy in
the 21st century: a sustainable development scenario.
International Journal of Hydrogen Energy 2003;28(3):267–84.
[6] Hallenbeck PC. Fundamentals of the fermentative
production of hydrogen. Water Science and Technology 2005;
52(1–2):21–9.
[7] Hallenbeck PC, Benemann JR. Biological hydrogen
production; fundamentals and limiting processes.
International Journal of Hydrogen Energy 2002;27(11–12):
1185–93.
[8] Nandi R, Sengupta S. Microbial production of hydrogen: an
overview. Critical reviews in microbiology 1998;24(1):61–84.
[9] Fascetti E, Todini O. Rhodobacter sphaeroides RV cultivation
and hydrogen production in a one- and two-stage chemostat.
Applied Microbiology and Biotechnology 1995;44(3–4):300–5.
[10] Markov T, Miura Y, Fukatsu K, Miyasaka H, Ikuta Y,
Matsumoto H, et al. Hydrogen production by photosynthetic
microorganisms. Applied Biochemistry and Biotechnology
1997;63-65(1):577–84.
[11] Fedorov AS, Tsygankov AA, Rao KK, Hall DO. Hydrogen
photoproduction by Rhodobacter sphaeroides immobilized
on polyurethane foam. Biotechnology Letters 1998;20(11):
1007–9.
[12] Levin DB, Pitt L, Love M. Biohydrogen production: prospects
and limitations to practical application. International Journal
of Hydrogen Energy 2004;29(2):173–85.
[13] Gustavo DV, Sonia A, Felipe AM, Antonio de LR, Luis MRC,
Elı́as RF. Fermentative biohydrogen production: trends and
819
perspectives. Reviews in Environmental Science and
Biotechnology 2008;7(1):27–45.
[14] Bisaillon A, Turcot J, Hallenbeck PC. The effect of nutrient
limitation on hydrogen production by batch cultures of
Escherichia coli. International Journal of Hydrogen Energy
2006;31(11):1504–8.
[15] Kawagoshi Y, Hino N, Fujimoto A, Nakao M, Fujita Y,
Sugimura S, et al. Effect of inoculum conditioning on
hydrogen fermentation and pH effect on bacterial
community relevant to hydrogen production. Journal of
Bioscience and Bioengineering 2005;100(5):524–30.
[16] Kraemer JT, Bagley DM. Continuous fermentative hydrogen
production using a two-phase reactor system with recycle.
Environmental Science and Technology 2005;39(10):3819–25.
[17] Lin CY, Cheng CH. Fermentative hydrogen production from
xylose using anaerobic mixed microflora. International
Journal of Hydrogen Energy 2006;31(7):832–40.
[18] Chen WM, Tseng ZJ, Lee KS, Chang JS. Fermentative
hydrogen production with Clostridium butyricum CGS5
isolated from anaerobic sewage sludge. International Journal
of Hydrogen Energy 2005;30(10):1063–70.
[19] Mu Y, Wang G, Yu HQ. Response surface methodological
analysis on biohydrogen production by enriched anaerobic
cultures. Enzyme and Microbial Technology 2006;38(7):905–13.
[20] Lee KS, Lin PJ, Chang JS. Temperature effects on biohydrogen
production in a granular sludge bed induced by activated
carbon carriers. International Journal of Hydrogen Energy
2006;31(4):465–72.
[21] Collet C, Adler N, Schwitzguebel JP, Peringer P. Hydrogen
production by Clostridium thermolacticum during
continuous fermentation of lactose. International Journal of
Hydrogen Energy 2004;29(14):1479–85.
[22] Ren NQ, Li J, Li B, Wang Y, Liu S. Biohydrogen production
from molasses by anaerobic fermentation with a pilot-scale
bioreactor system. International Journal of Hydrogen Energy
2006;31(15):2147–57.
[23] Yasuda K, Tanisho S. Fermentative hydrogen production
from artificial food wastes. In: Proceedings of the 16th world
hydrogen energy conference. Lyon, France; 2006. p. 210.
[24] Vijayaraghavan K, Ahmad D, Bin Ibrahim MK. Biohydrogen
generation from jackfruit peel using anaerobic contact filter.
International Journal of Hydrogen Energy 2006;31(5):569–79.
[25] Vijayaraghavan K, Ahmad D. Biohydrogen generation from
palm oil mill effluent using anaerobic contact filter.
International Journal of Hydrogen Energy 2006;31(10):
1284–91.
[26] Shin HS, Youn JH, Kim SH. Hydrogen production from food
waste in anaerobic mesophilic and thermophilic
acidogenesis. International Journal of Hydrogen Energy 2004;
29(13):1355–63.
[27] Kim SH, Han SK, Shin HS. Feasibility of biohydrogen
production by anaerobic co-digestion of food waste and
sewage sludge. International Journal of Hydrogen Energy
2004;29(15):1607–16.
[28] Han SK, Shin HS. Biohydrogen production by anaerobic
fermentation of food waste. International Journal of
Hydrogen Energy 2004;29(6):569–77.
[29] Lay JJ, Lee YJ, Noike T. Feasibility of biological hydrogen
production from organic fraction of municipal solid waste.
Water Research 1999;33(11):2579–86.
[30] Okamoto M, Miyahara T, Mizuno O, Noike T. Biological
hydrogen potential of materials characteristic of the organic
fraction of municipal solid wastes. Water Science and
Technology 2000;41(3):25–32.
[31] Park HS, Kim BH, Kim HS, Kim HJ, Kim GT, Kim M, et al. A
novel electrochemically active and Fe(III)-reducing bacterium
phylogenetically related to Clostridium butyricum isolated
from a microbial fuel cell. Anaerobe 2001;7(6):297–306.
820 international journal of hydrogen energy 34 (2009) 812–820
[32] Yokoi H, Tokushige T, Hirose J, Hayashi S, Takasaki Y. H2
production from starch by a mixed culture of Clostridium
butyricum and Enterobacter aerogenes. Biotechnology Letters
1998;20(2):143–7.
[33] Ting CH, Lin KR, Lee DJ, Tay JH. Production of hydrogen and
methane from wastewater sludge using anaerobic
fermentation. Water Science and Technology 2004;50(9):223–8.
[34] Cheong DY, Hansen CL. Acidogenesis characteristics of
natural, mixed anaerobes converting carbohydrate-rich
synthetic wastewater to hydrogen. Process Biochemistry
2006;41(8):1736–45.
[35] Sylvia DM, Fuhrmann JJ, Hartel PG, Zuberer DA. Principles
and applications of soil microbiology. Englewood Cliffs, NJ:
Prentice Hall; 1999.
[36] Setlow P. Resistance of bacterial spores. In: Storz G, Hengge-
Aronis R, editors. Bacterial stress responses. Washington,
DC: ASM Press; 2000. p. 217–30.
[37] Lay JJ. Modeling and optimization of anaerobic digested
sludge converting starch to hydrogen. Biotechnology and
Bioengineering 2000;68(3):269–78.
[38] Van Ginkel S, Sung S, Lay JJ. Biohydrogen production as
a function of pH and substrate concentration. Environmental
Science and Technology 2001;35(24):4726–30.
[39] Fan Y, Li C, Lay JJ, Hou H, Zhang G. Optimization of initial
substrate and pH levels for germination of sporing hydrogen-
producing anaerobes in cow dung compost. Bioresource
Technology 2004;91(2):189–93.
[40] Lee YJ, Miyahara T, Nioke T. Effect of pH on microbial
hydrogen fermentation. Journal of Chemical Technology and
Biotechnology 2002;77(6):694–8.
[41] Cheong, DY. Studies of high rate anaerobic bio-conversion
technology for energy production during treatment of high
strength organic wastewaters. PhD Thesis. Logan, Utah: Utah
State University; 2005.
[42] Ministry of Environmental Protection of the P. R. China.
China Environmental State Bulletin:2006, http://www.sepa.
gov.cn/plan/zkgb/06hjzkgb;; 2006.
[43] Salerno MB, Park W, Zuo Y, Logan BE. Inhibition of
biohydrogen production by ammonia. Water Research 2006;
40(6):1167–72.
[44] APHA. Standard method for the examination of water and
wastewater. New York: American Public Health Association;
1995.
[45] Lee YJ, Miyahara T, Noike T. Effect of iron concentration on
hydrogen fermentation. Bioresource Technology 2001;80(3):
227–31.
[46] Logan BE, Oh SE, Kim IS, van Ginkel S. Biological hydrogen
production measured in batch anaerobic respirometers.
Environmental Science and Technology 2002;36(11):2530–5.
[47] Oh SE, van Ginkel S, Logan B. The relative effectiveness of pH
control and heat treatment for enhancing biohydrogen gas
production. Environmental Science and Technology 2003;
37(22):5186–90.
[48] Chen CC, Lin CY, Lin MC. Acid-base enrichment enhances
anaerobic hydrogen production process. Applied
Microbiology and Biotechnology 2002;58(2):224–8.
[49] Zheng XJ, Yu HQ. Inhibitory effects of butyrate on biological
hydrogen production with mixed anaerobic cultures. Journal
of Environmental Management 2005;74(1):65–70.
[50] Mu Y, Wang G, Yu HQ. Kinetic modeling of batch hydrogen
production process by mixed anaerobic cultures. Bioresource
Technology 2006;97(11):1302–7.
[51] Lay JJ. Biohydrogen generation by mesophilic anaerobic
fermentation of microcrystalline cellulose. Biotechnology
and Bioengineering 2001;74(4):280–7.
[52] Gottschalk G. Bacterial metabolism. 2nd ed. New York:
Springer; 1986.
[53] Zoetemeyer RJ, Matthijse AJCM, Cohen A, Boelhouwer C.
Product inhibition in the acid forming stage of the anaerobic
digestion process. Water Research 1982;16(5):633–9.
[54] Switzenbaum MS, Giraldo-Gomez E, Hickey RF. Monitoring of
the anaerobic methane fermentation process. Enzyme
Microbial Technology 1990;12(10):722–30.
[55] Thauer R, Jungerman K, Decker K. Energy conservation in
chemotrophic anaerobic bacteria. Bacteriological. Reviews
1977;41(1):100–80.
[56] Heyndrickx M, Vos PD, Vancanneyt M, Ley JD. The
fermentation of glycerol by Clostridium butyricum LMG
1212t2 and 1213t1 and C. pasteurianum LMG 3285. Applied
Microbiology and Biotechnology 1991;34(5):637–47.
[57] Hanaki K, Matsuo T, Nagase M. Mechanism of inhibition
caused by long-chain fatty acids in anaerobic digestion
process. Biotechnology and Bioengineering 1981;23(7):
1591–610.
[58] Nagase M, Matuo T. Interactions between amino-acid
degrading bacteria and methanogenic bacteria in anaerobic
digestion. Biotechnology and Bioengineering 1982;24(10):
2227–39.
[59] Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS. Microbial
cellulose utilization: fundamentals and biotechnology.
Microbiology and Molecular Biology Reviews 2002;66(3):
506–77.